Evidence of a role for interleukin-6 in anoikis resistance in oral squamous cell carcinoma

In an endeavour to understand metastasis from oral squamous cell carcinomas, we characterised the metastatic potential of a human tongue derived cell line (SCC-4 cells) and compared this phenotype to pre-cancerous dysplastic oral keratinocyte (DOK) cells derived from human tongue and primary gingival keratinocytes (PGK). We demonstrate that SCC-4 cells constitutively synthesize and release significant amounts of IL-6, a process that is enhanced by the addition of the TLR2/TLR6 agonist, Pam2CSK4. The expression of TLR2/6 and IL-6Ra/gp130 receptors was also confirmed in SCC-4 cells. Cancerous SCC-4 human tongue cells also have a classic EMT profile, unlike precancerous human tongue DOK cells. We also established that IL-6 is driving anoikis resistance in an autocrine fashion and that anti-IL-6 neutralising antibodies, anti-IL-6 receptor antibodies and anti-TLR2 receptor antibodies inhibit anoikis resistance in cancerous SCC-4 human tongue cells. The data suggest a promising role for anti-IL-6 receptor antibody and anti-TLR2 receptor antibody treatment for oral cancer. Supplementary Information The online version contains supplementary material available at 10.1007/s12032-022-01664-5.


Introduction
Oral squamous cell carcinomas (OSCC) are the sixth most common type of cancer in the world and accounts for more than 90% of oral malignancies [1]. OSCC are metastatic cancers and arise from several anatomic sites within the oral cavity but most commonly from the tongue. Chemokines and cytokines (mostly interleukins) have been found to play key roles in cancer metastasis by, promoting the detachment and survival (anoikis resistance) of cancer cells from the primary tumour sites, affecting the epithelial to mesenchymal transition (EMT), regulating cancer cell migration and stimulating proliferation [2][3][4]. Normal cells undego apoptosis when they lose adhesion to the extracellular matrix (ECM), a process termed as "anoikis", from the Greek phrase "without home". Metastatic cancer cells develop the ability to survive and grow detached from the extracellular matrix and are thus termed anoikis resistant [5]. Cytokines IL-6 and IL-8 have been demonstrated to synergistically regulate a range of characteristics of cancer metastasis which were found to cooperatively regulate cancer cell proliferation, migration and the seeding of circulating tumour cells in various cancer cell types [6][7][8]. In addition, autocrine synthesis of IL-6 has been observed in a wide range of malignant tumours, including OSCC [9][10][11]. Furthermore, there is evidence that autocrine IL-6 production is an important element in tumour metastasis and enhancement of cell migration and invasion in human chondrosarcoma and osteosarcoma [12][13][14]. It is also noteworthy that anoikis resistance observed in pancreatic cells, was further increased upon IL-6 treatment [15]. In general, tumour metastasis to distant organs is a crucial step in establishing the aggressive phenotype of human cancer and it usually results in mortality of cancer patients [16]. TLR2, a member of the trans-membrane toll-like receptors family, is expressed on the surface of many cell types, including various cancer cell types and it is activated by several reported ligands [17][18][19]. Recent observations by Dong et al. [20] indicated a correlation between upregulated TLR2 expression and increased cell proliferation, invasion and migration of colorectal cancer. Additionally, inhibition of TLR2 signalling was demonstrated to suppress colorectal cancer cell growth, revealing a potential key role of TLR2 in colorectal tumorigenesis [20]. In this study we investigated TLR2 ligand-dependent IL-6 release and IL-6-dependent anoikis resistance in OSCC.

DOK and SCC-4 cell lines
Dysplastic oral keratinocyte (DOK) cells were originally isolated from a piece of dorsal tongue of a 57-year-old male. DOK cells [cat# ECACC 94122104] are characterized as Caucasian derived epithelial adherent tongue dysplastic cells.
Squamous cell carcinoma (SCC-4) cells were originally established from the tongue of a 55-year-old male [SCC-4 cat# ECACC 89062002]. Mycoplasma-free cells were grown in cell culture flasks in Dulbecco's Modified Eagle's Medium GlutaMAX cell culture medium (Gibco) supplemented with 5 µg/ml hydrocortisone, 20% (v/v) Foetal Bovine Serum (FBS) and penicillin-streptomycin (50 U/ml and 50 μg/ml) (Gibco). Cells were grown at 37 °C in humidified environment containing 95% O 2 and 5% CO 2 . DOK and SCC-4 cells were passaged at least twice weekly depending on their levels of confluency (75-80%) and were purchased from the European Centre of Authenticated Cell Culture. In terms of validity of comparability, all cells used in this study originated from the human buccal cavity and represent primary (PGK), pre-cancerous (DOK) and cancerous (SCC4) cells, with the latter two being from the same tissue (tongue).

Viability assay
AlamarBlue ® (Invitrogen) was employed to quantitatively measure the viability of cells. Resazurin, the active cellpermeable ingredient of alamarBlue ® , has a blue colour and after entering the cells, is reduced to resorufin, which then produces a red fluorescence. Viable cells are able to convert resazurin to resorufin giving a quantitative measure of their viability, while non-viable cells with a worse innate metabolic activity produce less resorufin. Plates were read on a Spectramax Gemini Plate Reader using SOFTmax Pro version 4.9 (Molecular Devices, Sunnyville, C.A, U.S.A.) at excitation and emission wavelengths of 544 nm and 590 nm, respectively. Experiments were conducted in triplicate and RFU values were expressed as mean ± SEM of the three experiments.

Measurement of cytokine concentration by ELISA
Commercially available ELISA kits were used according to manufacturer's instructions. Once sufficient colour was developed, 20 μl/well stop solution (2 N H 2 SO 4 ) was added and OD values were obtained by measure absorbance at 450 nm using Spectramax Microplate Reader (Molecular Devices). Concentrations of cytokines were determined using the standard curve from each ELISA plate. Commercial Human ELISA kit information: IL-6, (Biolegend/ MSC); IL-8 (Biolegend/MSC); TNF-α (Biolegend/MSC); IL-11 (R&D Systems).

Protein determination using the bicinchoninic acid (BCA) assay
Quantification of protein concentrations in cell lysates was carried out using the Bicinchoninic Acid (BCA) Assay as described by Smith et al. [21].

Densitometry
Densitometric analyses of protein expression on western blots were performed using the Image Lab Software, Bio-Rad Laboratories.

Statistical analysis
Statistical analyses were performed using the computer based mathematical package Graph Pad Prism 8.0 software. All results were expressed as mean ± standard error of the mean (SEM). For comparisons of two groups data were analyzed using a two-tailed unpaired student's t-test, while for comparisons of more than two groups, data were analyzed using one-way or two-way ANOVA followed by Tukey's or Sidak's or Bonferroni's multiple comparison tests were performed. For all comparisons, p-value of *p < 0.05, **p < 0.01, ***p < 0.001 were considered to be significant.

Effect of anti-TLR2 blocking antibody on cytokine production and TLR2/TLR6 protein expression levels in DOK and SCC-4 cells
Having established the optimal concentration of anti-TLR2 blocking antibody in DOK and SCC-4 cells (Supplemental Fig. S2), the effect of anti-TLR2 antibody (10 μg/ml) on IL-6 release was analysed in these cell lines (Fig. 2). DOK ( Fig. 2A) and SCC-4 ( Fig. 2B) cells were pre-treated with 10 μg/ml anti-TLR2 blocking antibody for 1 h prior to stimulation with TLR2 agonists (Pam2CSK4 and Pam3CSK4). Measurement of IL-6 production after 24 h demonstrated that anti-TLR2 significantly inhibits IL-6 secretion in SCC-4 cells, while no IL-6 release was detected in DOK cells in the groups treated with anti-TLR2 antibody or in combination with TLR2 ligands. In particular, anti-TLR2 blocking antibody significantly diminished IL-6 secretion (461 ± 73.9 pg/ml (3)) when it was administered alone, while when combined with Pam2CSK4, IL-6 secretion decreased (794.8 ± 134.1 pg/ml (3)) significantly compared to Pam2CSK4 treated group (3026 ± 34.7 pg/ml (3)) but not significantly different when compared to the untreated control (1162 ± 18.2 pg/ml (3)). Interestingly, there was no effect of Pam3CSK4 alone or in combination with anti-TLR2, on IL-6 production when compared to the untreated control for SCC-4 cells (mean ± SEM (n)).
Based on the aforementioned TLR2 ligand-dependent observations, immunoblot analysis was used to determine protein expression levels of TLR2 and TLR6 in both DOK and SCC-4 cells (Fig. 3A, B). Collated data revealed that TLR2 expression (Fig. 3C) is significantly higher in untreated SCC-4 cells compared to untreated DOK cells. Interestingly, no significant downregulation of TLR2 was observed when both cell lines were treated with anti-TLR2 or anti-TLR2 in combination with TLR2 ligands (Pam2CSK4 and Pam3CSK4) when compared to their untreated controls. Furthermore, TLR6 expression ( Fig. 3D) is also significantly higher in untreated SCC-4 compared to DOK cells, but there was no effect on TLR expression with addition of anti-TLR2 blocking antibody alone or in combination with TLR ligands (Pam2CSK4 and Pam3CSK4) in either DOK or SCC-4 cells.

IL-6Ra and gp130 protein expression and effect of anti-TLR2 blocking antibody on IL-6 production in DOK and SCC-4 cells
Immunoblot analysis revealed that both gp130 and IL-6Ra are more prominent in SCC-4 cells compared to DOK cells. Figure 4A and B displays one representative immunoblot. Collated data in Supplemental Fig. S3 shows that (A) gp130 and (B) IL-6Ra expression is significantly greater in SCC-4 cells compared to DOK cells, and that rhIL-6 treatment had no effect on expression levels.

A comparison of epithelial to mesenchymal transition (EMT) profile of dysplastic oral keratinocytes (DOK) cells and immortal squamous cell carcinoma 4 (SCC-4) cells
In order to determine the EMT profile in DOK and SCC-4 cells, immunoblot analysis was employed and cells were examined untreated and treated with 30 ng/ml rhIL-6, 10 μg/ ml IL-6 monoclonal neutralising antibody, or 40 μg/ml IL-6Ra monoclonal antibody for 24 h. Cells were collected, lysed and ran on 8% SDS-PAGE gel before being transferred to PVDF membrane and probed using antibodies for E-cadherin, N-cadherin, β-catenin and vimentin. An anti-GAPDH antibody was used as a loading control. Both cell lines were treated with 10 μg/ml of an IgG isotype antibody for 24 h as a control for the IL-6 monoclonal neutralising antibody. Examples of these immunoblots for DOK cells are given in Supplemental Fig. S4A and B for SCC-4 cells and display one representative experiment of untreated and treated with 10 μg/ml IgG isotype control, 30 ng/ml rhIL-6, 10 μg/ml IL-6 neutralising monoclonal antibody and 40 μg/ml IL-6Ra monoclonal antibody. As shown significant upregulated β-catenin was found in untreated control SCC-4 cells compared to untreated control DOK cells (Fig. 5), while no Ε-cadherin was detected in SCC-4 cells (Supplemental Fig. S4C). Interestingly, both N-cadherin (Supplemental Fig. S4D) and vimentin (Supplemental Fig. S4E) were upregulated in SCC-4 cells, however no expression of the aforementioned proteins was shown in DOK cells respectively). Furthermore, no differences were detected in the expression of the proteins examined, regardless of the treatments received (rhIL-6, IL-6 monoclonal neutralising antibody and IL-6Ra monoclonal antibody) compared to the untreated and isotype controls in either DOK or SCC-4 cells. Lysates were collected and samples were run on 12% SDS-PAGE gel. Western Blot were probed with anti-TLR2 and anti-TLR6 antibodies with anti-GAPDH used as a loading control. Experiment was done in triplicate. Densitometric analysis was performed using image lab software on TLR2, TLR6 and GAPDH blots. Sample blots for (A) DOK and (B) SCC-4 and collated densitometry data for (C) TLR2 and (D) TLR6 are presented. Results were plotted using Graphpad prism 8. Values represent the mean ± S.E.M of three independent experiments. Statistical analysis was performed using two-way ANOVA with a post hoc Sidak's test to compare mean values among the groups, *p < 0.05; **p < 0.01; ***p < 0.001

Discussion
Of the cytokines investigated, IL-6 was the most abundant cytokine produced by our cancerous cells which led us to give it some focus in this study. It was demonstrated that IL-6 secretion is threefold higher from SCC-4 cells compared to normal PGK, while IL-6 secretion is undetectable from DOK cells. This observation is consistent with that of Chen et al. [25] who observed similar amount of elevated IL-6 production in SCC-4 cells compared to another oral squamous cell carcinoma, SCC-25 cells, which showed threefold lower IL-6 production. Another study [26] also suggested enhanced production of salivary IL-6 as a diagnostic marker for leukoplakia and OSCC cells. Furthermore, clinical studies have reported increased IL-6 serum levels and salivary IL-6 concentration in OSCC patients compared to patients with leukoplakia and healthy controls, suggesting that IL-6 could be a crucial biomarker in HNSCC diagnosis [27,28].
Our investigation progressed to determine whether TLR ligand activation had an effect on IL-6 production levels from the cells used in this study. When SCC-4 cells were treated with the TLR2/TLR6 agonist, Pam2CSK4, there was a significant threefold increase in IL-6 production in both SCC-4 and PGK cells. No significant increase in IL-6 production was observed with TLR1/TLR6 ligand, Pam3CSK4. Interestingly, Chuang et al. [29] examined the intracellular signalling pathway involved in IL-6-induced intracellular adhesion molecule-1 (ICAM-1) expression and tumour migration in SCC-4 cells by activating a number of crucial pathways, and a possible role for IL-6 in oral cancer metastasis was suggested. It is noteworthy that when SCC-4 cells were treated with the TLR4 agonist, LPS, IL-6 secretion levels significantly increased but not to the same extent as with TLR2/TLR6 agonist, Pam2CSK4. However, there is evidence in the literature that lipopolysaccharide (LPS) is able to induce TLR4-mediated epithelial-mesenchymal transition and cell migration in SCC-4 cells [30] which may or may not be due to IL-6.
By contrast, it was revealed that both PGK and DOK cells IL-11 secretion levels were threefold higher when compared to SCC-4 cells. Interestingly, only Pam3CSK4 and LPS agonists slightly but significantly affected IL-11 secretion levels in DOK cells, while only Pam3CSK4 significantly diminished IL-11 production in PGK cells. Significant IL-11 increase was also found in SCC-4 cells after stimulation with all agonists but in general IL-11 levels were significantly lower than the levels in treated dysplastic counterparts. Elevated levels of IL-11 (~ 250 μg/l) have been identified in breast cancer patients which is associated with bone metastasis [31].
TNF-α, a widely expressed pleiotropic cytokine, is increased during inflammation and cancer in saliva and patient sera. Indeed, it has been reported that TNF-α levels might act as diagnostic markers for detection of oral cancer since it is more abundant in saliva than in plasma [32,33] and Scheff et al. [34] demonstrated significant secretion of TNF-α from certain oral squamous cancer cells in a pain model. However, in our in vitro system, TNF-α secretion levels were undetectable regardless of the stimulations received in DOK and SCC-4 cells, while negligible detection of TNF-α production was observed in normal PGK cells.
IL-8 has been found in the tumour microenvironment in a range of different cancer types, including OSCC tumours. Clinical studies have reported elevated salivary IL-8 levels in OSCC patients [35,36] compared to healthy controls. However, the results in this study did not reveal substantial IL-8 production in SCC-4 cells.
TLRs have been reported to be expressed in various types of cancers with key roles in carcinogenesis and tumour progression [37]. Since Pam2CSK4, a TLR2/6 agonist, was the ligand that induced the most IL-6 secretion, and to a lesser extend IL-8 and IL-11 cytokine production in SCC-4 cells, TLR2 neutralising antibody combined with either Pam2CSK4 or Pam3CSK4 were used to further examine their effect on IL-6 production in DOK and SCC-4 cells. It was demonstrated that treatment with anti-TLR2 neutralising antibody resulted in a twofold decrease in IL-6 production from SCC-4 cells, while IL-6 was undetectable from precancerous DOK cells, and no effect of anti-TLR2 neutralising antibody could be detected. Furthermore, no effect on the IL-6 production was observed after treatment with TLR2 neutralising antibody combined with Pam3CSK4, which led us to postulate that TLR1 is probably not expressed to any great degree in either DOK or SCC-4 cells.
In support of these findings, a previous report revealed higher TLR2 expression in the microenvironment of the keratinocytes of dysplastic epithelium and OSCC, when compared to hyperplasic cells [38]. Interestingly, a report by Ikehata et al. [39] identified, by western blot and immunohistochemistry, enhanced expression of TLR2, TLR1 and TLR6 in human OSCC tissue compared to adjacent nonmalignant tissue, as well as significant amount of TLRs in a range of OSCC cells (HSC3, HSC3-M3, SCC-9, SCC-25 cells). The data in this study are consistent with these observations, in that TLR2 and TLR6 were found significantly upregulated in SCC-4 cells compared to DOK cells.
After observing the effect of TLR2/6 ligand on IL-6 secretion from DOK and SCC-4 cells and the levels of TLR2 expression in these cells, it was decided to investigate whether there was expression of receptors for IL-6, or more particularly expression of IL-6Ra and gp130, in DOK and SCC-4 cells using western blotting. These findings suggest significantly higher IL-6Ra and gp130 expression in SCC-4 cells compared to DOK cells, regardless of the stimulation with rhIL-6. This result suggests that classical IL-6 signaling occurs in SCC-4 cells since both receptors were expressed, and consequently IL-6 is able to bind to the membranebound receptor IL-6Rα, which has the capacity for heterodimerization with gp130 and thus forming a complex that allows the JAK/STAT signaling cascade to operate [40].
The effect of endogenous and exogenous IL-6 on cancer phenotypes of pre-cancerous human tongue DOK cells and cancerous SCC-4 human tongue cells were also investigated. Anoikis resistance, markers of EMT, and cell death were characterized in these cells. Evidence is provided for IL-6 playing a central role in anoikis resistance. Data from this study show that DOK cells displayed non-EMT characteristics, expressing E-cadherin and lower levels of β-catenin compared to SCC-4 cells. High levels of vimentin and N-cadherin were observed in SCC-4 cells compared to DOK cells, while complete loss of E-cadherin was demonstrated in SCC-4 cells, confirming the EMT phenotype of these cells. Moreover, it was demonstrated by Wu et al. [41] that IL-6 induced EMT-related gene expression, leading to EMT phenotypes in pancreatic cancer cell (AsPC-1, BxPC-3, and Panc-1 cells), in which mesenchymal-like markers including N-cadherin and vimentin were upregulated, and epitheliallike marker, E-cadherin were downregulated. Interestingly, E-cadherin was upregulated while N-cadherin and vimentin were downregulated upon treatment with IL-6 neutralising antibody in these pancreatic cells [41]. Similarly, decreased expression of E-cadherin and increased vimentin was observed in response to IL-6 in osteosarcoma cells (U2OS and MG-63 cells), while those effects were reversed after treatment with small interferring-IL-6, suggesting a potential implication of IL-6 in acquisition/maintenance of stemness properties [42]. Another study has reported that activation of IL-6 signalling is correlated with aggressive tumour behaviour and EMT changes in pharyngeal cancer [43], lymph node metastasis and disease recurrence [25], while activated IL-6Ra/gp130 signalling induced various pathways associated with the regulation of tumour proliferation and metastasis [44]. However, in this study, there was no effect of rhIL-6 addition, addition of IL-6 neutralising antibody or addition of IL-6Ra monoclonal antibody on β-catenin, E-cadherin, N-cadherin or vimentin expression levels in either DOK or SCC-4 cells, suggesting that EMT profile is not regulated by IL-6 in these cells.
Anoikis resistance, or the ability for cells to live detached from the extracellular matrix, is a property of epithelial cancers. In this study, resistance to anoikis was assessed in DOK and SCC-4 cells. The results demonstrated that SCC-4 cells showed significantly greater resistance to anoikis when compared to DOK cells. In particular, half the population of untreated dysplastic DOK cells (49.2%) were able to resist anoikis when detached from the ECM, while survival rates of untreated cancerous SCC-4 cells were significantly higher (1.5-fold at 76.5%) as might be expected from cancerous cells. It was then decided to look at the effect of IL-6 on anoikis resistance in DOK and SCC-4 cells. The results indicated that rhIL-6 treatment significantly enhanced (1.4fold) resistance to anoikis in SCC-4 cells when compared to untreated control levels, an observation consistent with that seen by Fofaria and Srivastava [15] for pancreatic cancer cells.
Similarly, anoikis resistance in SCC-4 cells was increased (1.3-fold) upon stimulation with Pam2CSK4, an observation that is consistent with a role for endogenous IL-6 being important for anoikis, and consistent with the observation that Pam2CSK4 triples IL-6 secretion from SCC-4 cells. Again, consistent with a role for endogenously produced IL-6 in anoikis, is the observation that treatment of SCC-4 cells with IL-6 receptor (IL-6Ra) monoclonal antibody significantly reduced (2.5-fold) anoikis resistance, a result that is further endorsed by the observation that neutralising IL-6 or blocking TLR2 with the monoclonal antibodies also reduces anoikis resistance in SCC-4 cells (1.8-fold). Consistent with these observations is the observation that anoikis resistance was not affected when extrinsic IL-6 was added in combination with IL-6Ra addition. There was no effect of rhIL-6 addition, IL-6Ra addition, IL-6 neutralizing antibody, TLR2 blocking antibody or a combination of rhIL-6 plus IL-6RA on anoikis resistance in pre-cancerous dysplastic DOK cells.

Summary and conclusions
We show that a quantitatively significant amount of IL-6 is secreted by oral cancerous SCC-4 cells. In addition, the TLR2 ligand Pam2CSK4 enhances IL-6 secretion from SCC-4 cells and enhances anoikis resistance. The quantitatively large increase in IL-6 production from SCC-4 cells with addition of the Pam2CSK4, but not Pam3CSK4, is consistent with our data confirming the existence of TLR2/6 and IL-6Ra/gp130 receptors in SCC-4 cells. The fact that DOK cells do not produce IL-6 is consistent with the lack of IL-6Ra/gp130 receptors in DOK cells. The role of TLR2 on enhanced IL-6 production is further cemented by the observation that anti-TLR2 receptor blocking antibodies reduced, by half, the IL-6 production in SCC-4 cells and significantly reduces anoikis resistance. The central role played by IL-6 in anoikis in SCC-4 cells is also demonstrated by the observations that added recombinant IL-6 increases anoikis resistance, and that IL-6 neutralizing antibody and IL-6 receptor antibodies reduce anoikis resistance. This study provides evidence, for the first time, that IL-6 drives anoikis resistance in SCC-4 cells.